In vitro actomyosin motility in deuterium oxide

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Actomyosin interaction is crucial for muscle contraction and cellular motility.
• The role of solvent isotopes, like deuterium oxide (D2O), on actomyosin dynamics is not fully understood.
• Investigating D2O's effect provides insights into the molecular mechanisms of actin-myosin interaction.

Purpose of the Study:

• To examine the effect of deuterium oxide (D2O) on the actomyosin interaction in an in vitro motility assay.
• To elucidate the kinetic parameters influencing actin filament sliding velocity in D2O compared to H2O.

Main Methods:

• In vitro motility assay measuring actin filament velocities in D2O and H2O.
• Michaelis-Menten kinetics analysis of ATP concentration-dependent velocity.
• Stopped-flow spectrophotometry to measure ADP release rates from the acto-S1-ADP complex.

Main Results:

• Actin filament sliding velocity in D2O was approximately 60% of that in H2O at optimal conditions.
• Deuterium oxide (D2O) decreased the Km for actin-activated Mg-ATPase activity of myosin subfragment 1 (S1) from 50 microM to 33 microM.
• The rate constant for ADP release from the actomyosin-ADP complex was significantly slower in D2O (361 s⁻¹) compared to H2O (512 s⁻¹).

Conclusions:

• The reduced in vitro actin-myosin sliding velocity in D2O is attributed to a slower ADP release from the actomyosin-ADP complex.
• Deuterium oxide increases the affinity of actin for myosin in the presence of ATP.
• These findings highlight the sensitivity of actomyosin dynamics to isotopic solvent effects.